The objective of this proposal is to investigate the regulatory mechanisms governing ACTH release from the anterior pituitary gland. The present understanding of ACTH secretion has profited from investigations using large cell populations. The specific aim of this proposal is to refine this understanding still further by using powerful new techniques that analyze ACTH release at the level of single pituitary cells. I will establish the physiologic effects of a "suite" of ACTH regulators given individually or in combination. The capability to establish the number of activated ACTH cells among mixed cell populations has recently been provided with the introduction of reverse hemolytic plaque assays to endocrinology. In addition, the amount of ACTH released from activated pituitary cells in culture can also be estimated on a cell-by-cell basis. With pharmacologic probes I will attempt to characterize the contribution of three major intracellular pathways in ACTH release elicited by individual ACTH regulators and combinations of them. The intracellular messenger systems implicated in ACTH secretion include the cAMP-protein kinase A pathway, the calcium-calmodulin pathway, and the diacylglycerolprotein kinase C pathway. A fundamental role of calcium mobilization to trigger hormone secretion has long been accepted but the nature of this process is poorly understood. I propose to directly quantify cytosolic calcium in ACTH cells. Probes such as "quin 2" can be loaded into cells and dynamically register cytosolic calcium levels by fluorescing. In combination with state-of-the-art microscopes, equipped with an image enhancement and intensification capacity, it may possible to quantify fluorescent signals accompanying calcium mobilization in single activated pituitary ACTH cells loaded with quin 2. These studies may have profound implications for the understanding of hormone secretion from the anterior pituitary. Basic studies such as these will provide a foundation upon which the better management of the panorama of stress-related disorders will rest.